Activation of phosphatidylinositol 3′-kinase (PI 3-kinase) by growth factors and oncogenes has been implicated as a critical step in mitogenic signaling, cellular transformation and in the prevention of cell death (apoptosis), as described in Cantley et al, Cell 64:281-302 (1991), Kapeller and Cantley. Bioessays 16:565-76 (1994), and Stephens et al, Biochim BiophysActa 1179:27-75 (1993). PI 3-kinase consists of 85 kDa and 110 kDa subunits which associate with receptor tyrosine kinases, other receptors and intracellular signaling molecules in response to survival signals, treatment with growth factors or in normal or transformed cells. Blockade of PI 3-kinase function either by mutagenesis or with pharmacological inhibitors prevents mitogenic signaling and can enhance apoptosis by blocking the activation of Akt/Protein Kinase B. Further, two products of PI 3-kinase, PtdIns(3,4,5)P3 (PIP3) and PtdIns(3,4)P2, increase in cells treated with mitogenic stimuli, as shown by Hawkins, et al. Nature 358:157-910, (1992) and Klippel et al, Molecular and Cellular Biology 16:41174127 (1996). The products of PI 3-kinase are presumed to act as second messengers, as regulators of protein-protein interactions, or recruit other kinases that phosphorylate downstream effectors of PI3K signaling.
Thus, engagement of receptors on the surface of mammalian cells results in the activation of phosphatidylinositol 3-phosphate kinase (PI-3 kinase) and phosphorylation of inositol phospholipids on the cytoplasmic side of the membrane. The generation of phosphatidyl inositol (3,4,5) triphosphate (PIP3) by PI-3 kinase contributes to the activation of signaling pathways that drive cell proliferation and/or prevent apoptosis. Removal of the phosphate group from the D5 position of phosphoinositides by the hematopoietic-specific SH2-containing Inositol Polyphosphatase (SHIP) has been identified as an important negative feedback mechanism influencing cell activation and survival in the mammalian hematolymphoid compartment.
SHIP was originally identified based on its ability to bind Shc, Grb2, the FcγRIIB receptor, and by a gene-trapping approach. Through the use of in vitro assays, it was demonstrated that SHIP can remove the 5′-phosphate of PIP3 and inositol 1,3,4,5-tetrakisphosphate (IP4) suggesting that SHIP may counteract the activity of PI-3 kinase or prevent the sustained influx of Ca2+ into the cell. The tyrosine phosphorylation and membrane recruitment of SHIP in response to receptor stimulation has been demonstrated in a variety of transformed hematolymphoid cell lines. Following activation of hematopoietic cells, SHIP is recruited to the membrane for better access to key substrates. In addition, mounting genetic evidence indicates that SHIP plays an important role in vivo as a negative regulator of cell activation in B lymphoid cells, myeloid cells, and mast cells. For example, one study demonstrated that SHIP−/− mice, although viable and fertile, failed to thrive, displaying only a 40% survival rate by 14 weeks of age. Mortality was associated with extensive consolidation of the lungs resulting from infiltration of myeloid cells. Increased numbers of granulocytes-macrophage progenitors were observed in both the bone marrow and spleen. Helgason, CD et al. (1998) “Targeted disruption of SHIP leads to hemopoietic perturbations, lung pathology, and a shortened life span.” Genes Dev. 12(11):1610-20. In another study, SHIP−/− mast cells were found to be more prone to mast cell degranulation than SHIP−/+ or +/+ cells. Huber, M. et al (1998) “The src homology 2-containing inositol phosphatase (SHIP) is the gatekeeper of mast cell degranulation.” Proc. Natl. Acad Sci USA 95(19):11330-5. In a third study, SHIP−/− mice exhibited chronic hyperplasia of myeloid cells which resulted in splenomegaly, lymphadenopathy, and myeloid infiltration of vital organs. Further, neutrophils and bone marrow-derived mast cells from these mice were less susceptible to programmed cell death induced by various apoptotic stimuli or by growth factor withdrawal. Liu, Q. et al. (1999) “SHIP is a negative regulator of growth factor receptor-mediated PKB/Akt activation any myeloid cell survival.” Genes Dev. 13(7):789-91; Liu, Q. et al. (1998) “The inositol polyphoshate 5-phosphatase SHIP is a crucial negative regulator of B cell antigen receptor signalling.” J Exp Med 188(7):1333-42.
Together, these results demonstrate that SHIP is an important regulator of cellular responses in mature cells of certain hematopoietic lineages. The above studies were conducted with knockout mice using the traditional approach of neomycin replacement of exon I of the SHIP gene.
Inositol polyphosphate 5-phosphatases were the subject of U.S. Pat. No. 6,090,621 to Kavanaugh et al. “Signaling inositol polyphosphate 5-phosphatases (SIPS)”; PCT WO9710252A1 to Rohrschneider, L. R. “DNA encoding an SH2-inositol phosphatase, a SHC binding protein”; and PCT WO9712039A2 to Krystal, G. “SH2 containing inositol phosphatase.”
None of the aforementioned studies have identified a role for SHIP in NK (natural killer) cell function, nor have these studies identified a role for NK cells in graft-versus-hosts disease (GVHD). It would be advantageous for reasons disclosed and described below, to control the activity of SHIP. Methods for controlling SHIP activity, and the benefits and treatments that the instant invention provides in improving bone marrow and solid organ transplants, potentially abrogating marrow graft and solid organ rejection, together with means for screening for substances that modulate-SHIP activity, and more, are contained herein as will become apparent to one of skill in the art upon reading the following disclosure.